Immunogenicity of variable regions of hepatitis C virus proteins: selection and modification of peptide epitopes to assess hepatitis C virus genotypes by ELISA.

Rodríguez-López, Mario Henry; Riezu‐Boj, José I.; Ruiz, Marta; Berasain, Carmen; Civeira, M.P.; Prìeto, Jesús; Borrás‐Cuesta, Francisco

doi:10.1099/0022-1317-80-3-727

Cited by 23 publications

(26 citation statements)

References 58 publications

(7 reference statements)

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“…In this study, we demonstrated that the carboxyterminal domains II and III of NS5A are important for priming NS5A-specific humoral immune responses. It is known that this region of NS5A contains several human B cell epitopes (53,54). Our results show that a rNS5A protein was more immunogenic in priming NS5A-specific IgG Abs compared with NS5A as a DNA plasmid.…”

Section: Discussionmentioning

confidence: 52%

A Synthetic Codon-Optimized Hepatitis C Virus Nonstructural 5A DNA Vaccine Primes Polyfunctional CD8+ T Cell Responses in Wild-Type and NS5A-Transgenic Mice

Holmström

Pasetto

Nähr

et al. 2013

The Journal of Immunology

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The hepatitis C virus (HCV) nonstructural (NS) 5A protein has been shown to promote viral persistence by interfering with both innate and adaptive immunity. At the same time, the HCV NS5A protein has been suggested as a target for antiviral therapy. In this study, we performed a detailed characterization of HCV NS5A immunogenicity in wild-type (wt) and immune tolerant HCV NS5A-transgenic (Tg) C57BL/6J mice. We evaluated how efficiently HCV NS5A-based genetic vaccines could activate strong T cell responses. Truncated and full-length wt and synthetic codon-optimized NS5A genotype 1b genes were cloned into eukaryotic expression plasmids, and the immunogenicity was determined after i.m. immunization in combination with in vivo electroporation. The NS5A-based genetic vaccines primed high Ab levels, with IgG titers of >104 postimmunization. With respect to CD8+ T cell responses, the coNS5A gene primed more potent IFN-γ–producing and lytic cytotoxic T cells in wt mice compared with NS5A-Tg mice. In addition, high frequencies of NS5A-specific CD8+ T cells were found in wt mice after a single immunization. To test the functionality of the CTL responses, the ability to inhibit growth of NS5A-expressing tumor cells in vivo was analyzed after immunization. A single dose of coNS5A primed tumor-inhibiting responses in both wt and NS5A-Tg mice. Finally, immunization with the coNS5A gene primed polyfunctional NS5A-specific CD8+ T cell responses. Thus, the coNS5A gene is a promising therapeutic vaccine candidate for chronic HCV infections.

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Section: Discussionmentioning

confidence: 52%

A Synthetic Codon-Optimized Hepatitis C Virus Nonstructural 5A DNA Vaccine Primes Polyfunctional CD8+ T Cell Responses in Wild-Type and NS5A-Transgenic Mice

Holmström

Pasetto

Nähr

et al. 2013

The Journal of Immunology

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“…NS4B is amongthe HCV major immunogenic proteins and it is widely used in anti-HCV antibody detection in diagnostic serologic tests (6)(7)(8). Furthermore, since NS4B is a key protein in virus life cycle, it provides new opportunities for antiviral intervention against a leading cause of liver diseases worldwide (12).…”

Section: Discussionmentioning

confidence: 99%

“…Some of the diagnostically important antigenic epitopes have been found to reside within NS4B (6)(7)(8). There are two FDAapproved commercially available enzyme immunoassays (EIA) for screening anti-HCV antibodies: Abbott HCV EIA 2.0 and ORTHO HCV Version 3.0 enzymelinked immunosorbent assay (ELISA) Test System.…”

Section: Introductionmentioning

confidence: 99%

Analysis of Immumoreactivity of Heterologously Expressed Non-structural Protein 4B (NS4B) from Hepatitis C Virus (HCV) Genotype 1a

et al. 2015

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Background: Detection of hepatitis C virus specific antibodies is the initial step in chronic HCV diagnosis. HCV NS4B is among the most immunogenic HCV antigens and has been widely used in commercial Enzyme Immunoassays (EIA). Additionally, NS4B, a key protein in the virus replication, can be an alternative target for antiviral therapy. Objectives: Development of a new method for high-level expression and purification of NS4B coding region was the aim of the report. Materials and Methods: Viral RNA was purified from the serum of an HCV positive patient and NS4B coding region was amplified using nested RT-PCR. PCR products were cloned into pET102/D-TOPO expression vector and transformed into E. coli BL21. Induction was performed by adding 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) to the culture medium. Immunoreactivity of the purified recombinant proteins was evaluated by immunoblotting and indirect enzymelinked immunosorbent assay (ELISA). Results: The recombinant NS4B protein was expressed and its immunoreactivity was confirmed by ELISA and western blotting. Conclusions: The directional TOPO cloning provides an efficient and easy platform for heterologous expression of immunoreactive HCV NS4B.

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“…The detection rate of anti-NS5 (59.3%) is much lower than the detection rates of anti-C22 (92.1%) and C33 (88.1%) (11). Commercial HCV tests based upon proteins or peptides derived solely from HCV genotype 1 may be less effective for the detection of antibody against HCV of other genotypes (8,17,24). Dow et al (10) found that sera from donors infected with different genotypes showed different patterns of reactivity by the third-generation confirmatory assay.…”

mentioning

confidence: 99%

“…Dow et al (10) found that sera from donors infected with different genotypes showed different patterns of reactivity by the third-generation confirmatory assay. As more data are accumulated, it has become increasingly evident that further improvement of HCV diagnostic tests will rely on the careful selection of sequence variants of antigens capable of detecting antibodies against different HCV genotypes with equal efficiencies.The antigenic compositions of many HCV proteins have been studied by using recombinant proteins (19) and synthetic peptides (13,24,25,26). Studies of the major HCV antigenic regions showed that sequence heterogeneity has an impact on the antigenic properties of these regions (17,19).…”

mentioning

confidence: 99%

Antigenic Heterogeneity of the Hepatitis C Virus NS5A Protein

et al. 2002

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The effect of sequence variability between different types of hepatitis C virus (HCV) on the antigenic properties of the NS5 protein was studied by using recombinant proteins. A strong antigenic region was identified within the HCV NS5A protein at amino acids 2212 to 2313. Forty-five unique sequences encompassing this region were selected from GenBank and were compared to each other. The results of this analysis showed that the primary structure of this strong antigenic region is highly variable. Percent homology between different genotype sequences varied from 40.4 to 72.5%. Thirteen representative sequences from all six HCV genotypes were selected to design synthetic genes coding for this antigenic region. These genes were assembled by PCR from synthetic oligonucleotides and expressed in Escherichia coli as hybrid proteins with glutathione S-transferase. All 13 fusion proteins were purified from bacterial lysates and used to test a panel of anti-HCV positive sera (n ‫؍‬ 91) obtained from patients infected with HCV genotypes 1 through 6. All but two proteins immunoreacted with 62 to 93% of HCV anti-NS5-positive serum samples. Although a variable degree of genotype-specific antigenic reactivity was detected, only one protein demonstrated a noticeable preference to immunoreact with antibodies against the homologous HCV genotype. On the other hand, closely related proteins derived from the same subtype or genotype immunoreacted with significantly different efficiency with HCV antibodies. Thus, sequence variability has a profound effect on the antigenic properties of the NS5A immunodominant regions. This observation should be taken into consideration in the development of diagnostic tests for the efficient detection of anti-HCV activity in serum specimens.The hepatitis C virus (HCV) is associated with the vast majority of cases of posttransfusion hepatitis and sporadic non-A, non-B hepatitis worldwide (6). The HCV genome is a single-stranded positive RNA coding for a polyprotein of ϳ3,000 amino acids (aa) (7) which is processed into 10 viral proteins, namely, core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (14, 23). The HCV genome is very heterogeneous and has been classified into six genotypes (18).HCV serologic diagnosis is achieved by detecting specific antibody by enzyme immunoassays (EIAs) that use antigens derived from core, NS3, NS4, and NS5 proteins. Although screening assays have been effective in reducing the risk of posttransfusion hepatitis C, these assays do not detect anti-HCV activity in all patients with HCV infection. For example, the second-generation EIA (EIA-2) detects anti-HCV in ϳ90% of HCV-infected patients (2). In accordance with this observation, 3 to 10% of anti-HCV-negative donors transmitted HCV to their recipients (1, 3, 28). In addition, these assays are prone to false-positive results, especially noticeable in lowrisk populations (9). For example, approximately 40 to 50% of specimens from healthy blood donors which were positive by EIA-2 could not be confirmed by a supplemental...

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Immunogenicity of variable regions of hepatitis C virus proteins: selection and modification of peptide epitopes to assess hepatitis C virus genotypes by ELISA.

Cited by 23 publications

References 58 publications

A Synthetic Codon-Optimized Hepatitis C Virus Nonstructural 5A DNA Vaccine Primes Polyfunctional CD8+ T Cell Responses in Wild-Type and NS5A-Transgenic Mice

A Synthetic Codon-Optimized Hepatitis C Virus Nonstructural 5A DNA Vaccine Primes Polyfunctional CD8+ T Cell Responses in Wild-Type and NS5A-Transgenic Mice

Analysis of Immumoreactivity of Heterologously Expressed Non-structural Protein 4B (NS4B) from Hepatitis C Virus (HCV) Genotype 1a

Antigenic Heterogeneity of the Hepatitis C Virus NS5A Protein

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